1. Field of the Invention
The present invention relates to monoclonal antibodies for human TNFs (tumor necrosis factors), and, more particularly, to monoclonal antibodies for a human TNF which are useful as a medicine and ensure immunological purification and measurement of said human TNF.
2. Description of the Background Art
A lipopolysaccharide (LPS), when administered to a mouse, rabbit, or rat sensitized by Bacillus de Calmette Guerin (BCG), induces a serum factor which hemorrhagically necroses tumors. Carswell et al. named this factor "TNF" in 1975 [Proc. Nat. Acad. Sci., USA, 72. 3666 (1975)]. Macrophages proliferate in the spleen of a mouse sensitized with BCG but elapse by the administration of LPS. From this, TNF has been considered to be produced by macrophages. Recent studies have unveiled the fact that an in vitro LPS treatment of isolated macrophages induced a TNF activity in the supernatant of the macrophage culture broth, confirming that macrophages are TNF-producing cells. Several leukemia cells producing TNF have also been reported.
The characteristic of TNF destroying various cancer cells and inhibiting their growth, while exhibiting no such effects on normal cells, suggests its potential utility as a carcinostatic agent. The anti-tumor activity of TNF has been confirmed in experiments using a purified TNF derived from mice and rabbits.
The TNF activity has been measured using a fibroblast cell line, L929, having a strong sensitivity to a TNF. The TNF unit of activity is generally expressed as the amount which results in a 50% cytotoxicity of L929 cells in a culture plate.
The molecular weights of TNF produced by various animals as determined by a gel filtration analysis are reported to be 150,000 and 40,000-60,000 for TNF produced by mouse, 67,000 and 39,000 for TNF produced by rabbit, and 34,000-140,000 for TNF produced by human. According to molecular weight determination using SDS-PAGE, purified TNFs derived from rabbit and human are reported to be 17,000. TNFs having this molecular weight are termed "TNF-.alpha." or tumor necrosis factor-alpha. Natural TNFs are considered to exist as oligomers.
In 1984, the cloning of TNF-cDNA was successfully achieved using human myeloma leukemia cells "HL-60" which are a type of TNF-producing cell. This has ensured large-scale production of TNF using E. coli. The cDNA cloning revealed the fact that human TNF is constituted by 157 amino acids and has a long preceding polypeptide having 76 amino acids [D. Pennica et al., Nature, 312, 724-729 (1984)]. Very closely following this, the successful cloning of TNF chromosomal genes was also reported [T. Shirai et al., Nature, 313, 803-806 (1985)], and it was revealed that human TNF genes are composed of four exons.
The amino acid sequence of TNF and the gene base sequence have respectively 28% and 46% homologies with a cytotoxin factor and a lymphotoxin (LT) produced by B-cells. TNF, however, has a substantive difference from LT in that the former has no N-glycosil-type sugar chain bonding site. Although TNF and LT have no immunological cross-reactivity, they exhibit very similar cytotoxicity. The homological portions of TNF and LT are considered to be involved in the cytotoxicity.
As mentioned above, because of its special physiological activity, the utility of TNF as a medicine can be expected. A number of studies have been undertaken with a view to utilize TNF as a medicine. Also, extensive research is are underway on the subject of various immunodeficiencies and immunoreactions, as well as on the subject of TNF assay in clinical samples of diseases related to immunodeficiencies and immunoreactions.
A bioassay technique is currently used for the analysis of the aforementioned TNF-.alpha.. According to this method TNF is measured in terms of its activity. The method, however, has problems in its complicated procedure and insufficient accuracy. The method also requires stringent precaution concerning any components interfering with the assay result. Development of a measurement technique substituting for the bioassay technique has therefore been sought.